Project Abstract While studies of gap junctions often cite their fundamental importance to normal electrical activation of the heart, there is little agreement on the degree of gap junction remodeling required to slow electrical activation. Our prior work has made the following seminal observations. First, the degree of cardiac hydration can modulate whether gap junction uncoupling will be associated with measurable conduction slowing and increased arrhythmia risk. Second, the microdomain adjacent to gap junction plaques is rich in the cardiac sodium channel isoform Nav1.5, is nominally 150 nm long and 10-20 nm wide. Through the use of super- resolution microscopy, electron microscopy, optical mapping, and cutting edge computations simulations, we provided evidence that the perinexus is a strong candidate structure as the cardiac ephapse. Third, we demonstrate that the buffer used to perform ex vivo whole-heart studies can modulate both perinexal width and the relationship between cardiac conduction and gap junctions. In short, altering extracellular sodium and potassium can compensate for a 50% loss of connexin43 such that conduction appears normal. These findings are entirely consistent with the various groups that have analyzed the relationship between conduction velocity and gap junctions, so much so that it is possible by post-hoc literature analysis to determine whether a group will find conduction slowing secondary to loss of Cx43 simply by the buffer used. This work is not just important for understanding alternative modes of electrical communication, but it points out an even more sobering issue: the foundational tool of ex vivo and in vitro biology (buffers) may yield investigator dependent results simply based on ionic buffer composition. In this application we will test three new aims. 1. We will test unique computational predictions of gap junctional and ephaptic coupling to demonstrate that the conduction reserve hypothesis is unique to continuous ?cable-like? propagation, and ephaptic coupling can self-attenuate conduction when intercellular widths are very narrow. 2. We will demonstrate how changing interfibrillar and perinexal separation separately alters cardiac conduction. These findings will be compared to models of ephaptic coupling in order to further support the hypothesis that ephaptic coupling is an alternative form of electrical coupling between myocytes. 3. We provide evidence that altering perfusate ion concentration can rescue conduction to the same degree as gap junction based therapy during acidosis. In this final aim, we will determine how perfusate composition modulates electrical coupling during no-flow ischemia and complete coronary artery ligation. Also, we will test whether perfusate composition can agonize the effects of gap junction therapy during ischemia.